Title: Purification of P-TEFb, a Transcription Factor Required for the Transition into Productive Elongation
Abstract: Production of full-length runoff transcripts <i>in vitro</i> and functional mRNA <i>in vivo</i> is sensitive to the drug 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB). We previously proposed the existence of an activity, P-TEF (positive transcription elongation factor) that functions in a DRB-sensitive manner to allow RNA polymerase II elongation complexes to efficiently synthesize long transcripts (Marshall, N. F. and Price, D. H.(1992) <i>Mol. Cell. Biol.</i> 12, 2078-2090). We have fractionated nuclear extracts of <i>Drosophila melanogaster</i> K<sup>c</sup> cells and identified three activities, P-TEFa, factor 2, and P-TEFb, that are directly involved in reconstructing DRB-sensitive transcription. P-TEFb is essential for the production of DRB-sensitive long transcripts <i>in vitro</i>, while P-TEFa and factor 2 are stimulatory. P-TEFb activity is associated with a protein comprising two polypeptide subunits with apparent molecular masses of 124 and 43 kDa. Using a P-TEFb-dependent transcription system, we show that P-TEFb acts after initiation and is the limiting factor in the production of long runoff transcripts.